1. Gene Regulation in Bacteria

2. INTRODUCTION
The term gene regulation means that the level of gene expression can vary under different conditions
Genes that are unregulated are termed constitutive
They have essentially constant levels of expression
Frequently, constitutive genes encode proteins that are continuously necessary for the survival of the organism
The benefit of regulating genes is that encoded proteins will be produced only when required
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3. Gene regulation is important for cellular processes such as
1. Metabolism
2. Response to environmental stress
3. Cell division
Regulation can occur at any of the points on the pathway to gene expression
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

4. TRANSCRIPTIONAL REGULATION
The most common way to regulate gene expression in bacteria is at the transcription initiation level
The rate of RNA synthesis can be increased or decreased
Transcriptional regulation involves the actions of two main types of regulatory proteins
Repressors  Bind to DNA and inhibit transcription
Activators  Bind to DNA and increase transcription
Negative control refers to transcriptional regulation by repressor proteins
Positive control to regulation by activator proteins
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

5. Regulatory proteins have two binding sites
One for a small effector molecule
The other for DNA

6. Copyright ©The McGraw-Hill Companies, Inc
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

7. Activator/Repressor and the effector molecules

8. Small effector molecules affect transcription regulation
However, these bind to regulatory proteins and not to DNA directly
In some cases, the presence of a small effector molecule may increase transcription
These molecules are termed inducers
They function in two ways
Bind activators and cause them to bind to DNA
Bind repressors and prevent them from binding to DNA
Genes that are regulated in this manner are termed inducible
In other cases, the presence of a small effector molecule may inhibit transcription
Corepressors bind to repressors and cause them to bind to DNA
Inhibitors bind to activators and prevent them from binding to DNA
Genes that are regulated in this manner are termed repressible
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9. The lac Operon
An operon is a regulatory unit consisting of a few structural genes under the control of one promoter
It encodes polycistronic mRNA that contains the coding sequence for two or more structural genes
This allows a bacterium to coordinately regulate a group of genes that encode proteins with a common function
An operon contains several different regions
Promoter; terminator; structural genes; operator
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

10. Copyright ©The McGraw-Hill Companies, Inc
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11. organization and transcriptional regulation of the lac operon genes
There are two distinct transcriptional units
1. The actual lac operon
a. DNA elements
Promoter  Binds RNA polymerase
Operator  Binds the lac repressor protein
CAP site  Binds the Catabolite Activator Protein (CAP)
b. Structural genes
lacZ  Encodes b-galactosidase
Enzymatically cleaves lactose and lactose analogues
Also converts lactose into allolactose (an isomer)
lacY  Encodes lactose permease
Membrane protein required for transport of lactose and analogues
lacA  Encodes transacetylase
Covalently modifies lactose and analogues
Its functional necessity remains unclear
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12. Organization and transcriptional regulation of the lac operon genes
There are two distinct transcriptional units
The lacI gene
Not considered part of the lac operon
Has its own promoter, the i promoter
Constitutively expressed at fairly low levels
Encodes the lac repressor
The lac repressor protein functions as a tetramer
Only a small amount of protein is needed to repress the lac operon
There is usually ten tetramer proteins per cell
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

13. The lac Operon Is Regulated By a Repressor Protein
The lac operon can be transcriptionally regulated
1. By a repressor protein
2. By an activator protein
The first method is an inducible, negative control mechanism
It involves the lac repressor protein
The inducer is allolactose
It binds to the lac repressor and inactivates it
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

14. RNA pol cannot access the promoter
Constitutive expression
The lac operon is now repressed
Therefore no allolactose
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

15. The lac operon is now induced Some gets converted to allolactose
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

16. The cycle of lac operon induction and repression
Repressor does not completely inhibit transcription
So very small amounts of the enzymes are made
The cycle of lac operon induction and repression
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

17. The lac Operon Is Also Regulated By an Activator Protein
The lac operon can be transcriptionally regulated in a second way, known as catabolite repression
When exposed to both lactose and glucose
E. coli uses glucose first, and catabolite repression prevents the use of lactose
When glucose is depleted, catabolite repression is alleviated, and the lac operon is expressed
The sequential use of two sugars by a bacterium is termed diauxic growth
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

18. The lac Operon Is Also Regulated By an Activator Protein
The small effector molecule in catabolite repression is not glucose
This form of genetic regulation involves a small molecule, cyclic AMP (cAMP)
It is produced from ATP via the enzyme adenylyl cyclase
cAMP binds an activator protein known as the Catabolite Activator Protein (CAP)
Also termed the cyclic AMP receptor protein (CRP)
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

19. The lac Operon Is Also Regulated By an Activator Protein
The cAMP-CAP complex is an example of genetic regulation that is inducible and under positive control
The cAMP-CAP complex binds to the CAP site near the lac promoter and increases transcription
In the presence of glucose, the enzyme adenylyl cyclase is inhibited
This decreases the levels of cAMP in the cell
Therefore, cAMP is no longer available to bind CAP
Transcription rate decreases
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

20. Copyright ©The McGraw-Hill Companies, Inc
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

21. Copyright ©The McGraw-Hill Companies, Inc
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

22. The trp Operon
The trp operon (pronounced “trip”) is involved in the biosynthesis of the amino acid tryptophan
The genes trpE, trpD, trpC, trpB and trpA encode enzymes involved in tryptophan biosynthesis
The genes trpR and trpL are involved in regulation
trpR  Encodes the trp repressor protein
Functions in repression
trpL  Encodes a short peptide called the Leader peptide
Functions in attenuation
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

23. RNA pol can bind to the promoter Cannot bind to the operator site
Organization of the trp operon and regulation via the trp repressor protein

24. Organization of the trp operon and regulation via the trp repressor protein

25. Organization of the trp operon and regulation via the trp repressor protein

26. The first gene in the trp operon is trpL
Attenuation occurs in bacteria because of the coupling of transcription and translation
During attenuation, transcription actually begins but it is terminated before the entire mRNA is made
A segment of DNA, termed the attenuator, is important in facilitating this termination
In the case of the trp operon, transcription terminates shortly past the trpL region Thus attenuation inhibits the further production of tryptophan
The segment of trp operon immediately downstream from the operator site plays a critical role in attenuation
The first gene in the trp operon is trpL
It encodes a short peptide termed the Leader peptide
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

27. Region 2 is complementary to regions 1 and 3
Therefore several stem-loops structures are possible
The 3-4 stem loop is followed by a sequence of Uracils
It acts as an intrinsic (r-independent) terminator
These two codons provide a way to sense if there is sufficient tryptophan for translation
Sequence of the trpL mRNA produced during attenuation

28. There are three possible scenarios
Therefore, the formation of the 3-4 stem-loop causes RNA pol to terminate transcription at the end of the trpL gene
Conditions that favor the formation of the stem-loop rely on the translation of the trpL mRNA
There are three possible scenarios
1. No translation
2. Low levels of tryptophan
3. High levels of tryptophan
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

29. Transcription terminates just past the trpL gene
Most stable form of mRNA occurs
Therefore no ribosome associated with the RNA
Possible stem-loop structures formed from trpL mRNA under different conditions of translation

30. Region 1 is blocked
3-4 stem-loop does not form
RNA pol transcribes rest of operon
Insufficient amounts of tRNAtrp
Possible stem-loop structures formed from trpL mRNA under different conditions of translation

31. Sufficient amounts of tRNAtrp
3-4 stem-loop forms
Translation of the trpL mRNA progresses until stop codon
Transcription terminates
Ribosome pauses on 2
Region 2 cannot base pair with any other region
Possible stem-loop structures formed from trpL mRNA under different conditions of translation

32. Inducible vs Repressible Regulation
The study of many operons revealed a general trend concerning inducible versus repressible regulation
Operons involved in catabolism (i.e. breakdown of a substance) are typically inducible
The substance to be broken down (or a related compound) acts as the inducer
Operons involved in anabolism (ie. biosynthesis of a substance) are typically repressible
The inhibitor or corepressor is the small molecule that is the product of the operon
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

33. TRANSLATIONAL AND POSTTRANSLATIONAL REGULATION
Genetic regulation in bacteria is exercised predominantly at the level of transcription
However, there are many examples of regulation that occur at a later stage in gene expression
For example, regulation of gene expression can be
Translational
Posttranslational
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

34. Translational Regulation
For some bacterial genes, the translation of mRNA is regulated by the binding of proteins
A translational regulatory protein recognizes sequences within the mRNA
In most cases, these proteins act to inhibit translation
These are known as translational repressors
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

35. Translational Regulation
Translational repressors inhibit translation in one of two ways
1. Binding next to the Shine-Dalgarno sequence and/or the start codon
This will sterically hinder the ribosome from initiating translation
2. Binding outside the Shine-Dalgarno/start codon region
They stabilize an mRNA secondary structure that prevents initiation
Translational repression is also found in eukaryotes
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

36. Translational Regulation
A second way to regulate translation is via the synthesis of antisense RNA
An RNA strand that is complementary to mRNA
Consider, for example, the trait of osmoregulation
The ability to control the amount of water inside the cell
Must be regulated to prevent shrinkage or lysis
The protein ompF in E. coli is important in osmoregulation
This outer membrane protein is encoded by the ompF gene
OmpF protein is preferentially produced at low osmolarity
At high osmolarity its synthesis is decreased
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

37. Its translation is now blocked
The expression of another gene, termed micF, is responsible for inhibiting the ompF gene at high osmolarity
micF RNA does not code for a protein
It is, however, complementary to ompF mRNA
It is thus termed antisense RNA
Its translation is now blocked
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

38. Posttranslational Regulation
A common mechanism to regulate the activity of metabolic enzymes is feedback inhibition
The final product in a pathway often can inhibit the an enzyme that acts early in the pathway
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

39. Catalytic site  binds substrate
Enzyme 1 is an allosteric enzyme, which means it contains two different binding sites
Catalytic site  binds substrate
Regulatory site  binds final product of the pathway
If the concentration of product 3 becomes high
It will bind to enzyme 1
Thereby inhibiting its ability to convert substrate 1 into product 1
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

40. Posttranslational Regulation
A second strategy to control the function of proteins is by the covalent modification of their structure
Some modifications are irreversible
Proteolytic processing
Attachment of prosthetic groups, sugars, or lipids
Other modifications are reversible and transiently affect protein function
Phosphorylation (–PO4)
Acetylation (–COCH3)
Methylation (–CH3)
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

43. Allosteric protein